The present invention relates generally to molecular biology and more specifically to a method for identifying protein pairs involved in proteinxe2x80x94protein interactions.
The phrase xe2x80x9cproteinxe2x80x94protein interactionxe2x80x9d refers to the ability of two protein molecules to bind to each other so as to form a complex. Such proteinxe2x80x94protein interactions are involved in a large variety of biological processes including, for example, signal transduction pathways, enzyme-substrate interactions, viral adhesions and the formation of antibody-antigen complexes.
Many proteins are capable of interacting with a number of other proteins. Identifying and characterizing such interactions are highly important in understanding biological mechanisms, signal transduction pathways, etc. in characterizing the molecular basis of various diseases as disorders and in the is design of therapies.
A defect in a protein preventing it from participating in a proteinxe2x80x94protein interaction can. as may be appreciated, have deleterious effects on a cell.
The ability to identify and characterize proteinxe2x80x94protein interactions permits the identification of the defects in such interactions associated with a diseased state. The identification of such defects provides a target for potential therapies to cure or ameliorate the disease. In addition, the identification and characterization of proteinxe2x80x94protein interactions provides a means to screen for drugs that alter the interaction. Such drugs can be useful, for example, to treat a disease caused, at least in part, by an aberrant proteinxe2x80x94protein interaction.
Methods for assaying proteinxe2x80x94protein interactions have been reviewed in Allen et al., Trends Biochem. Sci., 20:511-516, 1995.
Proteins involved in a proteinxe2x80x94protein interaction can be identified by detecting the presence of the protein complex in a cell or in a body fluid, and purifying the proteins forming the complex by biochemical methods. Such methods of isolation, however, are extremely tedious, particularly when the protein is expressed at low levels or if only a few cells express the protein. Immobilization of proteins on membrane filters has more recently lead to the development of filter based assays using proteins translated from cDNA molecules obtained, for example, from phage. However, the filter based assays, while being more sensitive, are also often very tedious.
A genetic method of identifying proteinxe2x80x94protein interactions has also been developed (Fields et al, Nature, 340:245-246, 1989). In this method, known as the xe2x80x9ctwo hybrid assayxe2x80x9d, one protein is fused to a DNA binding domain (typically from the Gal4 protein) while another protein is fused to a strong transcription activation domain. Binding of the two proteins inside a cell thus generates a functional transcription factor that is detected by a change in phenotype of the cell due to the expression of genes whose transcription is under the control of Gal4 DNA elements. The two hybrid system however, suffers from several limitations. First, protein pairs in which one of the proteins possesses transcriptional activity on its own, obviously cannot be analyzed. This includes bona fide transcription factors as well as proteins containing domains that fortuitously interact with the transcription machinery. Another limitation of the two hybrid system results from the toxicity of many proteins, for example certain homeodomain proteins and cell cycle regulators, when expressed in the nucleus. Furthermore, the two hybrid system produces false positive or false negative results when one of the proteins undergoes a conformational chance in the nucleus.
Another genetic method, the xe2x80x9cSos Recruitment Systemxe2x80x9d (SRS) has also been described (Aronheim, A., Mol Cell. Biol., 17:3094-3102, 1997). This method is based on the observation that localization of the protein hSos (the Ras guanyl nucleotide exchange factor) at the plasma membrane is essential for activating the Ras pathway and is therefore essential for viability. A yeast strain, such as cdc25-2, containing a temperature sensitive allele of Cdc25, (a yeast homologue of hSos) is thus viable only at the permissive temperature (24xc2x0 C.). In the SRS system, a first protein (the bait protein) is fused to hSos while a second protein (the prey protein) is fused to a membrane localization domain. A proteinxe2x80x94protein interaction between the bait and prey proteins localizes hSos at the plasma membrane. This complements the Cdc25 mutation which is detected as cell growth at the restrictive temperature (36xc2x0 C.). However, the SRS also exhibits several limitations. First, about 20-30% of all bait proteins fused to hSos result in prey-independent complementation of Cdc25, a fact which yields a relatively high unspecific background signal (xe2x80x9cnoisexe2x80x9d). Another limitation of the SRS system is that the effector part of the hSos is relatively large (150 Kda). This tends to complicate the fusion to hSos of both large bait proteins as well as short bait proteins.
Another problem of the SRS system is due to the fact that Ras encoded proteins are able to bypass the Cdc25 mutation because the yeast GTPase activating proteins (IRA genes) hydrolyze GTP bound to mammalian Ras proteins rather inefficiently thus leaving the Ras proteins in their active GTP-bound form.
The present invention makes use of the fact that in order for it to function. Ras needs to be localized at the plasma membrane. This localization normally occurs via the covalent attachment of a lipid moiety to cysteine 186 that anchors Ras at the membrane. Ras contains a consensus CAAX box located at the C-terminal end which undergoes famesylation and subsequently palmitoylation. A Ras lacking the famesylation box (CAAX) is non-functional since it cannot be localized at the membrane. The present invention thus makes use of cells with a Ras that is mutated such that it cannot be localized at the membrane, e.g. lacking the farnesylation box or having a mutation therein. These cells are xe2x80x9cengineeredxe2x80x9d such that they express two fusion proteins, one fusion protein comprising a first protein (referred to herein at times as the xe2x80x9cbaitxe2x80x9d) and a Ras protein which is mutated such that it cannot bind to the plasma membrane, and another fusion protein which comprises a second protein (referred to herein at times as the xe2x80x9cpreyxe2x80x9d) and a membrane localization domain. If the bait binds the prey then the Ras fused to the prey becomes localized at the membrane and can thereby function.
FIG. 1 shows a schematic representation of the invention. In panel A, a cell incapable of expressing a functional Ras is made to express a Ras that cannot be localized at the membrane (and is thus non-functional) fused to a bait protein. A putative prey protein has been localized at the plasma membrane. A proteinxe2x80x94protein interaction between the prey and bait proteins (panel B) localizes Ras at the plasma membrane. This produces a functional Ras that is detected as a phenotypic change in the cell.
This Ras Recruiting System (RRS) has several advantages over the SRS system:
1. The Ras protein is relatively small, thereby overcoming several of the technical limitations and practical problems posed by the large size of Sos as described above.
2. The RRS system exhibits substantially less false positive results, as compared to the SRS, with mammalian cDNA expression library screens and therefore represents a more efficient system for characterizing interacting proteins.
The invention thus provides a method for identifying a proteinxe2x80x94protein interaction between a first protein and a second protein comprising the steps of:
(a) expressing in a cell which is incapable of activating a Ras protein;
(aa) a first nucleic acid sequence encoding a first fusion protein, said first fusion protein comprising a Ras protein mutated such that it cannot localize at the cell membrane and does not require an exchange factor fused to said first protein; and
(ab) a second nucleic acid sequence encoding a second fusion protein said second fusion protein, comprising said second protein fused to a plasma membrane localization domain; and
(b) determining whether there is a phenotypic expression of a functional Ras protein in said cell, the presence of a functional Ras protein in said cell indicating a proteinxe2x80x94protein interaction between said first protein and said second protein.
In a preferred embodiment of the invention, the mutated Ras protein, which forms part of the fusion proteins encoded by the first nucleic acid sequence, lacks a farnesylation box.
In another preferred embodiment of the invention, the cell incapable of expressing a functional Ras is of the yeast strain cdc 25-2. The Ras of this cell is non-functional at the restrictive temperature (36xc2x0 C.) due to a lack of a functional guanyl nucleotide exchange factor. Production of a functional Ras in these cells by an interaction between a bait protein and a prey protein according to the invention is detected as growth independent of a functional exchange factor at such a restrictive temperature, e.g. at about 33-37xc2x0 C., typically at about 36xc2x0 C.
The method of the invention is useful for screening of gene libraries to fixed expression products that interact with a specific protein. As already pointed out above determining proteinxe2x80x94protein interaction may be highly important for drug development. In addition, determining such interaction may serve as a diagnostic trend; for example, a specific pattern of interaction of one protein with others, may serve as an indication of a normal or a mutated protein.
The method of the invention lends itself also to application in high throughput screening techniques. Cells may be automatically supplemented with DNA constructs, e.g. plasmids, under conditions in which such constructs will be internalized by the cells and then screened automatically for such with a Ras phenotype expression.
The invention also provides a system for use in determining whether a first protein is capable of binding to a second protein, comprising:
(a) a culture of cells incapable of activating a Ras protein;
(b) a first nucleic acid vector, for inserting therein a DNA sequence encoding a first fusion protein which comprises a Ras protein mutated such that it cannot localize to the cell membrane and does not require an exchange factor and said first protein;
(c) a second nucleic acid vector, which may be the same or different than said first nucleic acid vector, for inserting therein a DNA sequence encoding a second fusion protein which comprises said second protein and a plasma membrane localization domain;
(d) reagents and devices for transfecting the cells with said first and said second nucleic acid;
(e) a monitoring arrangement for monitoring phenotypic Ras expression in said cells.
Also provided by the invention is a kit comprising some or all of the constituents of the above system.
Localization of a mammalian Ras fused to a bait of interest at the plasma membrane through a proteinxe2x80x94protein interaction in a temperature sensitive mutant such as cdc25-2 permits growth at 36xc2x0 C. Ras localization can also complement a temperature sensitive mutant yeast strain that is defective in its exchange factor. hSos, however, complements the yeast cdc25 mutant strain only when expressed in the membrane, but Rasts fails to do so in the yeast Rasts mutant strain, which is defective in its Ras. This eliminates isolation of mammalian Ras exchange factors in a library screen.
As provided by the invention, mammalian expression vectors comprising regions encoding for interacting protein partners used in the yeast RRS system can be co-tansfected with a reporter gene such as a chloramphenicol acetyl transferase (CAT) or luciferase gene under the control of either AP-1 responsive elements or Ras responsive elements. Cultured mammalian cells expressing these plasmids allow a proteinxe2x80x94protein interaction known to occur in yeast to be quantitatively detected in mammalian cells by monitoring the enzymatic activity of the reporter gene following the proteinxe2x80x94protein interaction and recruitment of activated Ras to the plasma membrane. Use of reporter genes in mammalian cells allows direct evidence of a proteinxe2x80x94protein interaction initially identified in yeast and improved assessment of drug effectiveness directly in mammalian cells.
The invention also provides a positive control for proteins for which a protein partner is not detected in a library screening as described above. In cells not expressing a functional Ras, a fusion protein is expressed comprising a functional Ras fused to two bait proteins. The first bait (xe2x80x9cthe tester baitxe2x80x9d) is the protein having no known protein partners while the second bait (xe2x80x9cthe control baitxe2x80x9d) is a protein having a known protein partner. The ability of the control bait moiety to bind its known prey is determined. A proteinxe2x80x94protein reaction occurring between the control bait moiety and its prey demonstrates that the gene for the fusion moiety is expressed at adequate levels that the Ras moiety of the fusion protein is functional, and that the Ras pathway is intact. This would provide genetic evidence that screening with the tester bait is potentially possible and worthwhile.
The fusion of a tester bait and a control bait to a single Ras molecule may be used for mapping the amino acids involved in an interaction between the tester bait and its prey. The tester bait is subjected to random mutagenesis and inserted fused in fame with the control bait. The tester bait DNA is inserted in frame with that of Ras and the control bait and expressed, for example, in cdc25-2 cells of one mating type. The cells are then mated with cells of the opposite mating type expressing either the prey of the tester bait or the control bait. Cells able to grow with the control bait but not with the tester bait prey indicate the integration of a mutation that affects the tester bait prey binding that is not due to a frame shift or nonsense mutation.
Similarly this embodiment may also be used for screening drugs for the ability to inhibit an interaction between a tester bait and its prey while not inhibiting an interaction between the control bait and its prey.